Inorganic polyphosphate (poly P), a polymer of hundreds of phosphate residues linked by high-energy bonds, is found in every cell in Nature-bacteria, fungi, plants and animals. Among poiy P functions are: kinase donor to glucose and nucleoside diphosphates, phosphate (P1) reservoir, divalent metal (Ca2+, Mg2+, Mn2+) chelator and component of a membrane complex in bacterial transformation. Our recent studies have disclosed three major roles: 1) "alarmone" in response to stresses and deficiencies, 2) adaptations for survival in the stationary phase and 3) virulence in some pathogens. Thus, poly P has a variety of functions depending on the cell and circumstances. The multiplicity of functions and the plasticity of microbial organisms has complicated the search for the biochemical mechanisms responsible for each action. The proposed research will focus on mechanisms of metabolic regulation. Based on promising preliminary studies, we intend to pursue two major lines: 1) Regulation of protein synthesis and turnover by the supply of amino acids by biosynthesis and protein turnover, and 2) Operations of phosphate uptake and efflux and concomitant divalent metal ion transport. In addition, we will be mindful of control of in vitro transcription of genes known to be activated by poly P in vivo and a possible role for poly P as a donor for protein kinases. The widespread conservation of the enzyme, poly P kinase (PPK), responsible for poiy P synthesis in bacteria, including many of the major pathogens, has led us to knockout ppk in several of these pathogens. In Pseudomonas aeruginosa poly P is essential for motility, quorum sensing, bioflim formation and virulence in mice. We expect that the proposed biochemical studies will reveal mechanisms that will assist in the current search for antimicrobial drugs aimed at PPK as a target. The research may also provide clues to the biosynthesis and metabolism of poly P in eukaryotes about which little is known. In view of the ubiquity of poly P in animal cells and subcellular organelles (particularly nuclei), it seems likely that poly P serves multiple functions in human metabolism that affect growth, differentiation and disease.